PROJECT ABSTRACT Tsg101 is a component of cellular ESCRT-I (endocytic sorting complex required for transport-I) and plays a well-established role in budding of HIV-1 and other pathogens from infected cells. In the case of HIV-1, the interaction occurs through a Pro-Thr-Ala-Pro (PTAP) motif in the HIV-1 structural precursor polyprotein, Gag. Ubiquitin (Ub) is also an important participant in Gag recognition. Translating these critical virus-host protein-protein interactions into useful therapeutic targets would have broad impact on anti-viral drug development strategy. Initially, random screening of a library of small molecules currently in use or in development as drugs for other indications was used to identify small molecules capable of blocking the interaction of Gag with Tsg101 without affecting cellular Tsg101 functions. We solved the NMR structure of Tsg101 in complex with a promising lead compound and now propose to improve the lead and discover additional small molecule inhibitors by conducting structure-activity relationship analyses (SAR) and computer-aided structure-based design. Innovative computational tools developed by our team for targeting protein-protein interfaces will be used to exploit the unique energetic and structural information inherent to molecular footprints (interaction maps) made by the initial lead, as well as PTAP, and Ub proteins in their respective binding interfaces within the Ub E2 variant (UEV) domain on Tsg101. The goal is identify small molecules compatible with the interfaces through virtual screening of large commercially available ligand libraries. It is anticipated that targeting Tsg101 rather than virus-encoded gene products as is currently done will circumvent several challenging problems associated with management of HIV infections, including resistant virus emergence, and potentially provide even wider impact through broad-spectrum application to other human pathogens requiring Tsg101 for replication. The specific goals are: (Aim 1) To conduct experimental SAR analyses and validation strategies on a recently identified targetable region in the Tsg101 UEV domain; (Aim 2) To conduct computational SAR and validation strategies that permit identification and refinement of new inhibitors using validated computational tools based on molecular footprints of PTAP, Ub, and the lead in their respective UEV binding pockets.